This P01 Program Project application seeks to develop insights into mechanisms by which antibodies (Abs) protect against HIV infection to facilitate design of improved Abs and effective immunogens. Development of effective vaccines or delivered Abs to control infection will require understanding of Ab interactions with antigen and with Ab receptors that mediate effector functions. Using knowledge of what Env mutations arise in response to HIV infection in humanized mice allows structural/bioinformatic analyses of which features promote Ab evasion, required information for designing broadly neutralizing antibodies (bNAbs) that are insensitive to common routes of viral evasion. This knowledge will allow optimization of the breadth/potency of bNAbs for passive delivery (both by injection and gene therapy reverse vaccination) and is required for effective immunogen design for vaccines, thus our project is relevant to both traditional and reverse vaccine strategies to combat HIV. To accomplish these goals and to establish basic principles underlying Ab-mediated protection, we will combine the expertise of the Nussenzweig, Ravetch, and Bjorkman laboratories in characterization of HIV bNAbs and humanized mouse models of HIV infection, antibody effector function evaluation and improvement, and the structural biology of Ab-HIV and Ab-receptor interactions. Our proposal comprises three separate, but inter-related and inter-dependent collaborative projects, with the following aims: (1) Test designed bNAbs in a humanized mouse model of HIV infection, sequence resistant HIV strains, evaluate bNAbs for ability to control established HIV infection in humanized mice, and evaluate novel immunogens in a mouse model; (2) Investigate the contributions of Fc effector function to HIV bNAbs in vitro and in vivo, including in a new in vivo mouse model for HIV entry and an AAV-based reverse immunization model in humanized mice; (3) Determine structural correlates of broad/potent neutralization and improved effector functions by solving crystal structures of designed and natural bNAbs complexed with HIV Env proteins and Fc receptors; design and test immunogens for eliciting bNAbs. These projects will be supported by an administrative core and three scientific cores comprising a cell/biochemical automation core to perform automated in vitro HIV neutralization and plate-binding assays, a protein expression core to express and purify recombinant proteins required for functional and structural studies, and an animal services core to generate/maintain mice required for in vivo experiments.